Neodecanoic acid, iron salt

Endpoint summary

Description of key information

The potential of Neodecanoic acid, iron salt to induce skin irritation (OECD 439, 431) and eye irritation (OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the eye. By assessing the results from both in vitro skin irritation/corrosion tests in a weight-of-evidence approach, the target substance must be considered as irritating to the skin (Skin Irrit. 2, H315).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-08-30 to 2017-10-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

- Storage: 2 to 8°C protected from light, under nitrogen
- Batch Number: LN11013894
- Retest Date : 31 July 2019
- Purity: 100%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

Human skin model, comprising a reconstructed epidermis with a functional stratum corneum

Vehicle:

other: The test article was administered without dilution

Details on test system:

Study Design:
In order to assess the potential non-specific reduction of the test article, 50 µL of test article was added to 1 mL of 1.0 mg/mL MTT and the colour change was assessed after incubation for 60 minutes at 37 ± 1 °C, 5 ± 1% CO2, 95% RH. There was no change in colour therefore the test article did not interact with MTT. 50 µL of test article was added to 0.3 mL deionized water and 0.3 mL isopropanol and incubated for 60 minutes at 37 ± 1 °C, 5 ± 1% CO2, 95% RH. The isopropanol solution become coloured, therefore it was deemed to have the potential to stain the tissue and additional controls were included in the assay. On the day of receipt EpiDermTMtissues were transferred to refrigerator at 2 to 8 °C and stored overnight. The next day, approximately 1 hour before starting the assay, the tissues were prepared for treatment in labelled 6-well plates. The test was performed on a total of four tissues per test article, negative and positive control, out of which two were used for a 3 minute application and two tissues were used for a 1 hour application. A volume of 50 µL of the undiluted test article was applied to the tissue. Further tissues were concurrently treated with 50 µL distilled water (negative control) and with 50 µL 8N potassium hydroxide (positive control). After the 3 minute or 1-hour contact periods, the tissues were washed with phosphate buffered saline (PBS) to remove residual material. The rinsed tissues were kept in 24-well plates (holding plates) until all tissues were dosed and rinsed. The positive control material is a direct MTT reducer, therefore additional positive controls were performed. The positive control article was applied to two freeze-killed tissues (thawed on the day of treatment) per exposure time. Once all tissues had been rinsed, they were transferred to wells containing 300 µL of 1 mg/mL MTT and were incubated for 3 hours (37 ± 1 °C, 5 ± 1% CO2, 95% RH). The additional control tissues were incubated with medium instead of MTT solution.
After incubation any resultant colour was extracted with 2 mL isopropanol. The plates were sealed and left at room temperature overnight. The optical density of each resultant extract was determined spectrophotometrically at 570 nm and cell viability was calculated for each tissue as a percentage of the mean of the negative control tissue. Skin corrosivity potential of the test article was classified according to the remaining cell viability obtained after test article treatment with either of the two treatment times.

Control samples:

other: A volume of 50 µL of the undiluted test article was applied to the tissue. Further tissues were concurrently treated with 50 µL distilled water (negative control) and with 50 µL 8N potassium hydroxide (positive control).

Amount/concentration applied:

A volume of 50 µL of the undiluted test article was applied to the tissue. Further tissues were concurrently treated with 50 µL distilled water (negative control) and with 50 µL 8N potassium hydroxide (positive control).

Duration of treatment / exposure:

3 and 60 min

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The OD values for the negative controls met the acceptance criteria, with the exception that the mean OD value for the three minute treatment was just below the range of 0.8 to 2.8 specified in the protocol. However, the result was within the laboratory historical control range of 0.486 to 2.154 and it was considered that the experiment was valid. Skin viability after the one hour exposure to the positive control article was <15%. The CV between the replicates should be =30%.

Interpretation of results:

GHS criteria not met

Conclusions:

The test article was considered to be non-corrosive according to the UN GHS classification system.

Executive summary:

This study was conducted according to OECD test guideline 431 to determine whether the test article causes corrosion in the in vitro skin model EpiDerm^TM. Duplicate EpiDerm^TM inserts were treated with test article, purified water (negative control) and 8N potassium hydroxide (positive control) for 3 minutes and 1 hour. At the end of the treatment period, the tissues were washed with phosphate buffered saline (PBS) and cell viability was assessed using the MTT assay. The skin corrosivity potential was classified according to the remaining cell viability obtained after test material treatment with either of the two treatment times. Skin viability was 86% after three minute and one hour exposures to the test article. The OD values for the negative controls met the acceptance criteria, with the exception that the mean OD value for the three minute exposure was just below the range of 0.8 to 2.8 specified in the protocol. Skin viability after a three minute or one hour exposure to the positive control article was 8% and 10%, respectively, demonstrating an acceptable performance of the assay. The test article was considered to be non-corrosive according to the UN GHS classification system.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-17 to 2018-02-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage: 2 to 8 °C under nitrogen, protected from light.
- Batch Number: LN11013894
- Retest Date: 31 July 2019
- Purity: 100%

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The study was conducted to meet the known requirements of Method B46 of Commission Regulation (EC) No 761/2009 and OECD Guidelines for Testing of
Chemicals Method 439 (adopted 28 July 2015).

Vehicle:

unchanged (no vehicle)

Remarks:

The test article was administered without dilution.

Details on test system:

EpiDermTM SIT (EPI-200) three-dimensional human skin model, comprising a reconstructed epidermis with a functional stratum corneum, supplied by MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic.

Control samples:

other: Three tissues per test article, negative control and positive control were treated by application of 30 µL of the negative control, 30 µL of the positive control and 30 µL of liquid test material onto the surface of the tissues.

Amount/concentration applied:

30 µL of liquid test material onto the surface of the tissues.

Duration of treatment / exposure:

The treated tissues were placed into an incubator at 37 ± 1 ºC, 5 ± 1% CO2 for 35 minutes. The plates were removed from the incubator and placed into a sterile
hood until the 60 minute treatment period was complete for each tissue. Following treatment, substances were removed by washing the tissues. The tissues were then placed on the appropriate medium and incubated for 41 hours.

Duration of post-treatment incubation (if applicable):

41 hours

Number of replicates:

Three tissues per test article, negative control and positive control

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

group mean

Value:

8.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

The group mean viability for the positive control was 3.8%.

Remarks on result:

positive indication of irritation

Remarks:

The group mean viability for the test article was 8.7%.

Other effects / acceptance of results:

The assay criteria were met.

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item, was considered to be irritant (UN GHS category 1 or 2) in the in vitro skin model EpiDermTM SIT (EPI-200).

Executive summary:

This study was conducted in accordance with OECD test guideline 439 to determine whether the test article causes irritation in the in vitro skin model EpiDermTM SIT (EPI-200). EpiDermTM SIT (EPI-200) inserts were treated with the test item, negative control (phosphate buffered saline (PBS)) and positive control (5% w/v sodium dodecyl sulphate (SDS)) for 60 minutes. At the end of the treatment period, the tissues were washed with PBS and cell viability was assessed using the MTT assay. The skin irritation potential was classified according to the remaining cell viability obtained after test article treatment. In the initial experiment, the negative control tissues failed to meet the required acceptance criteria and so the experiment was repeated. The first experiment is not reported further. In the repeat experiment, the group mean viability for the test article was 8.7% and for the positive control it was 3.8%, when compared to the negative control. Based on the results, the test item was considered to be irritant (UN GHS category 1 or 2) in the in vitro skin model EpiDermTM SIT (EPI-200).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-05 to 2017-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Storage: 2 to 8 °C, under nitrogen
- Batch Number: LN11013894
- Retest Date: 31 July 2019
- Purity: 100%

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.

- On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

- Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32 ± 1 °C for at least 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

- A volume of 750 µL of the test article was applied to each of three corneas.
- A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.

Duration of treatment / exposure:

A volume of 750 µL of the test article was applied to each of three corneas followed by a ten minute incubation at 32 °C. After this incubation period, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH change (demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red and the anterior chamber was filled with fresh MEM.
The corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber was emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 µL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer. A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.

Number of animals or in vitro replicates:

three corneas

Details on study design:

- Terminal Procedures: At the end of the assay all corneas were preserved in 10% Neutral Buffered Formalin. However, the Sponsor confirmed that microscopic examination was not required. The corneas were discarded on finalisation of the study report.

Irritation parameter:

cornea opacity score

Run / experiment:

mean

Value:

0.44

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

It was concluded that the test item produced an IVIS score of 0.44 and does not require classification for eye irritation.

Other effects / acceptance of results:

The assay was considered valid as the assay acceptance criteria were met.

Corneal Opacity

- The mean corrected opacity reading for the test article was 0.3.

- The mean corrected opacity reading for the negative control was 0.0.

- The mean corrected opacity reading for the positive control was 61.7.

- One cornea dosed with the test article was noted to be slightly cloudy after treatment. The corneas dosed with the positive control were noted to be cloudy and blistered after treatment.

Corneal Permeability

- The mean group corrected optical density for the test article was 0.007.

- The mean group corrected optical density for the negative control was 0.000.

- The mean group corrected optical density for the positive control was 0.394.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on an IVIS score of 0.44 obtained in the bovine corneal opacity and permeability assay (BCOP, OECD 437), the test item is considered as non-irritant to the eye.

Executive summary:

This study was conducted in accordance with OECD test guideline 437 to determine whether the test article causes serious eye damage or does not require classification for eye irritation, using the bovine corneal opacity and permeability (BCOP) assay. A mean in vitro irritation score (IVIS) of 0.44 was determined. The positive and negative controls induced the appropriate responses, indicating the validity of the assay. Based on the results, the test item can be considered as non-irritant to the eye and no classification is warranted.

 

               

               

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of Neodecanoic acid, iron salt to induce skin irritation (OECD 439, 431) and eye irritation (OECD 437) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the eye. By assessing the results from both in vitro skin irritation/corrosion tests in a weight-of-evidence approach, the target substance must be considered as irritating to the skin (Skin Irrit. 2, H315).

Justification for classification or non-classification

Based on the results, the target substance Neodecanoic acid, iron salt must be considered as irritating to the skin, but not to the eye. Thus, classification as Skin Irrit. 2, H315 is warranted in accordance to CLP Regulation 1272/2008.